Abstract: The methods and compositions disclosed herein relate to preparing libraries to sequence long molecules comprising repetitive regions. In some embodiments, this strategy involves substituting selected nucleotides in each molecule to predetermined, different nucleotides at low frequencies in random positions and co-barcoding the molecule in single-tube LFR reactions to generate mutagenized barcoded fragments. Each mutagenized barcoded fragment comprises a barcode, and the barcode comprises a barcode sequence.

Abstract: This application relates to methods and compositions for preparing a library of polynucleotides for sequencing comprises in a single reaction mixture. The method comprises contacting a double-stranded target nucleic acid with one or more nicking agents to produce overlapping nucleic acid fragments separated by staggered single-stranded breaks; and contacting a partially double-stranded first adapter with at least one of the nucleic acid fragments in the presence of a ligase, thereby ligating the 5? terminus of the double-stranded region of the first adapter to the 3? terminus of the at least one of the nucleic acid fragments using a DNA ligase via 3? branch ligation. The first adapter comprises (i) a double-stranded blunt end having a 5? terminus and a 3? terminus and (ii) a single-stranded region comprising a barcode.

Abstract: The invention provides novel 2D-VCAM-1 variant polypeptides and conjugates thereof that bind human VLA4. The invention also provides related polynucleotides, compositions, vectors, host cells, and methods.